The present invention is directed to methods for controlling cellular contraction and to medical treatments that rely upon this control. The methods are important in the treatment of ligament damage, osteoporosis, wound healing, tissue engineering, drug delivery, and the prevention of tumor cell metastasis.
Cellular contraction plays a role in a number of biological activities that have therapeutic consequences. The contraction of fibroblasts, grown on a matrix in vitro as part of a tissue engineering protocol for replacing a damaged ligament, may alter the size, shape, and porosity of the matrix and thereby jeopardize the performance of the implant. In vivo, inappropriate contraction may make the reattachment of the ends of a ruptured ligament to each other or to bone difficult or limit the movement of limbs as a result of excessive contracture. This scenario also applies to other musculoskeletal tissue cells and to epithelial cells such as endothelial cells and liver.
Cellular contraction also plays an important role in wound healing. Although contraction may initially promote healing, it can also lead to significant scarring and a loss of physiological function (see U.S. Pat. No. 5,741,777). The adverse effects of contraction are particularly severe in surgical and burn patients. In addition, scarring may cause secondary damage to patients that have incurred damage to the spinal cord or other severe trauma.
Other biological activities believed to depend, in part, on cellular contraction include osteoporosis (where the contraction of osteoblasts results in their retraction, thus allowing bone resorption to proceed) and in tumor cell metastasis. In the latter case, cancer cells must typically pass an endothelial cell barrier before they can enter into the bloodstream and be carried to a distant site for colonization. Agents that prevent endothelial cells from contracting (and thereby retracting) should therefore make metastasis more difficult. addition to its importance in ligament repair, tissue engineering, wound healing, osteoporosis and metastasis, the ability to control cell contraction may lead to improved procedures for drug delivery. For example, an agent that promoted endothelial cell contraction might be include in intranasal or intramuscular vehicles to aid in the passage of drug through the walls of capillaries. Such agents may also aid substances already in the bloodstream in exiting into tissue.
Alpha smooth muscle actin (SMA) is a particular isoform of actin that may cause the contraction of vascular smooth muscle cells (Saga, et al., Exp. Cell Res. 249:279-292 (1999)). It is known to be expressed in myofibroblasts during wound healing and in tissues undergoing fibrosis (Zhang, et al., Am. J. Pathol. 148:527-537(1996) Jester, et al., Opthal. Vis. Sci. 36:809-819(1995); Darby, et al., Lab. Invest. 63:21-28 (1990)). The complete nucleotide and amino acid sequences of the human form of the gene have been reported (Herrman, Curr. Opin. Cell Biol. 5:48-55 (1993)).
The present invention is based upon the discovery that SMA is responsible for the contraction of a variety of cells other than fibroblasts and for which such activity was not previously known. Agents that inhibit SMA activity prevent these cells from contracting, whereas agents that induce SMA activity promote contraction. Examples of SMA inhibitors include platelet derived growth factor (PDGF), staurosporin and interferons. An example of an SMA inducer is transforming growth factor-xcex2 (TGF-xcex2).
In its first aspect, the present invention is directed to methods of repairing musculoskeletal tissue (including bone, articular cartilage, meniscus, tendon intervertebral disk and especially damaged ligaments) and epithelial tissue. One procedure for accomplishing this involves removing cells from a patient""s body, growing them on a matrix, and then implanting the matrix/cell combination at the site of the damage, e.g. at the site of a tom ligament. This approach has been referred to as xe2x80x9ctissue engineering.xe2x80x9d The cells used may be any of the musculoskeletal, or epithelial cells mentioned above. Alternatively, marrow stromal stem cells may be used and have the advantage of being relatively easy to obtain. Matrices may be made out of several different types of biologically compatible material, but type I collagen and synthetic polymers, such as polylactic acid and polyglycolic acid, will typically be employed. The invention is directed to an improvement in this procedure in which cells grown on matrices in vitro are treated with a concentration of an agent sufficient to either inhibit or promote the expression or biological action of alpha-smooth muscle actin (SMA). Among the agents that maybe used for inhibiting contraction are PDGF and interferon. The activity of SMA may also be reduced by preventing its expression using an antisense oligonucleotide, particularly an oligonucleotide complementary to the promoter region of the human SMA gene. Among the agents that may be used to promote contraction is TGF-xcex2.
The invention is also directed to a method of treating a patient for damaged musculoskeletal tissue (particularly a damaged ligament), or epithelial tissue by sequentially administering, at the site of injury, an SMA inhibitor followed by an SMA inducer. The inhibitor should be given at a dosage and for a duration sufficient to promote tissue attachment. The time necessary for attachment to occur will vary from patient to patient, but will typically be between 1 and 10 weeks. The extent to which attachment has occurred may be determined by clinical examination and by diagnostic imaging techniques well known in the art. After attachment, the inducer should be administered for the purpose of causing the tissue to contract and thereby assume a more natural conformation. One example of a treatment protocol using this procedure would involve injections of TGF-beta at a concentration of between 100 ng/ml and 500 ug/ml at the site of ligament damage, e.g., the knee. After a period of, for example, 4 weeks, injections are made using a comparable concentration of PDGF or an interferon until healing is complete.
In another aspect, the invention is directed to a procedure for promoting the healing of wounded musculoskeletal tissue in a patient. Initially, an SMA inducer (TGF-beta, 100 ng/ml-500 ug/ml) is injected at the site of tissue damage at a dosage and for a duration sufficient to promote the closure of the wound. Once closure has been essentially completed, an SMA inhibitor (e.g., PDGF or an interferon in the concentration ranges recited above) may be administered at the site of the wound to reduce scar formation. In most cases, it is expected that administration will be accomplished using local delivery.
Inducers of SMA may also be administered to a patient for the purpose of enhancing drug absorption. A sufficient dosage should be given to induce endothelial cell contraction. For example, TGF-xcex2 at a concentration of 100 ng/ml-500 ug/ml can be co-administered with a second drug either parenterally or intranasally.
The invention is also directed to a method of preventing tumor cell metastasis in a cancer patient. This may be accomplished by administering an agent that inhibits SMA in the endothelial cells of the vasculature. Because the endothelial cells do not contract, cancer cells shed from a main tumor mass is prevented from entering into the patient""s bloodstream and those in the bloodstream are prevented from invading tissue.